Topological analysis of DPY19L3, a human C-mannosyltransferase

Yuki Niwa, Yoshihiko Nakano, Takehiro Suzuki, Mizuo Yamagishi, Kei Otani, Naoshi Dohmae, Siro Simizu

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase of R-spondin1 in human cells. DPY19L3 is predicted to be a multipass transmembrane protein that localizes to the endoplasmic reticulum (ER); however, its structure is undetermined. In this study, we propose a topological structure of DPY19L3 by in silico analysis and experimental methods such as redox-sensitive luciferase assay and introduction of N-glycosylation sites, suggesting that DPY19L3 comprises 11 transmembrane regions and two re-entrant loops with the N- and C-terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY19L3 has four predicted N-glycosylation sites, and we have demonstrated that DPY19L3 is N-glycosylated at Asn118 and Asn704 but not Asn319 and Asn439, supporting our topological model. By mass spectrometry, we measured the C-mannosyltransferase activity of N-glycosylation-defective mutants of DPY19L3 and isoform2, a splice variant, which lacks the C-terminal luminal region of DPY19L3. Isoform2 does not possess C-mannosyltransferase activity, indicating the importance of the C-terminal region; however, N-glycosylations of DPY19L3 do not have any roles for its enzymatic activity. These novel findings on DPY19L3 provide important insights into the mechanism of C-mannosylation.

Original languageEnglish
JournalFEBS Journal
DOIs
Publication statusAccepted/In press - 2018 Jan 1

Keywords

  • C-mannosylation
  • Glycosyltransferase
  • Multipass membrane protein
  • Re-entrant loop
  • Redox-sensitive luciferase assay

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Niwa, Y., Nakano, Y., Suzuki, T., Yamagishi, M., Otani, K., Dohmae, N., & Simizu, S. (Accepted/In press). Topological analysis of DPY19L3, a human C-mannosyltransferase. FEBS Journal. https://doi.org/10.1111/febs.14398